The present invention relates to a diagnostic reagent for detecting hepatitis C virus (HCV) infection by utilizing immunoagglutination.
In regard to hepatitis C, HCV gene was detected by the research group of Chiron Corporation, U.S.A., in 1988 prior to the detection of HCV. To detect antibodies to HCV, various recombinant antigens or synthetic peptides have been investigated, and kits for detecting HCV-associated antibodies have been developed. The methods of detection now available are agar diffusion, counterimmunoelectrophoresis, radioimmunoassay, enzyme immunoassay, passive hemagglutination, and latex agglutination.
Known HCV antigen proteins for use in the detection of HCV-associated antibodies are core and envelope proteins as structural region proteins, and NS1 to NS5 proteins as non-structural region proteins. One HCV antigen protein alone is not sufficiently high in detection sensitivity, and is also problematical in specificity. Thus, a suitable combination of proteins in structural and non-structural regions is used (Proc. Natl. Acad. Sci. USA 89:10011-10015, 1992). Attempts to increase the detection sensitivity further are also made. With the particle agglutination method, for example, the number of HCV antigens for sensitization of particles is increased, or polypeptide having HCV antigenic activity is heat-treated (Japanese Laid-Open Patent Publication No. 1002273/94); alternatively, a fusion protein constructed from HCV antigen protein and carrier protein is coated onto hydrophilic particles for their sensitization (Japanese Laid-Open Patent Publication No. 198723/95).
The use of synthetic peptide as an antigen has also been attempted, but this use is generally said to lower the detection sensitivity.
Thus, there is a growing demand for a diagnostic reagent and a method of diagnosis which enable many samples to be measured with high sensitivity and rapidity.
We have conducted intensive studies in an attempt to attain such objectives. As a result, we have found that high sensitivity can be realized by chemically bonding the core antigen, NS3 antigen, NS4 antigen or NS5 antigen of HCV to carrier protein by the glutaraldehyde method to form conjugated antigens, and sensitizing carrier particles with these conjugated antigens. The use of these conjugated antigens has also markedly improved the stability of the particles.
The present invention provides a diagnostic reagent for hepatitis C virus infection obtained by sensitizing a solid phase with HCV antigen and a conjugated antigen prepared by chemical bonding of HCV antigen and a carrier protein.
The carrier protein may be any water-soluble protein, preferably the one with a molecular weight of 10,000 to 1,000,000, more preferably with a molecular weight of 30,000 to 150,000. Preferred examples are bovine serum albumin (BSA), ovalbumin, and hemocyanin. In addition, water-soluble synthetic polymers, such as polyvinyl alcohol and dextran, are also usable.
The solid phase may be carrier particles, a microtiter plate, or a test tube, but carrier particles are preferred. As the carrier particles, known particles generally used in a diagnostic reagent involving the particle agglutination method can be used. Examples include hydrophobic particles such as polystyrene latex, copolymer latex particles having a hydrophilic group such as an amino or carboxyl group on the surface of the particles, erythrocytes, and gelatin particles. More preferable is polystyrene latex.
The HCV antigen protein used in the diagnostic reagent of the present invention is the known structural region protein or non-structural region protein of HCV. The structural region protein may be core protein, while the non-structural region protein may be NS3 protein, NS4 protein or NS5 protein. The amino acid and nucleotide sequences of these antigenic proteins are described in the literature (Officially Published Patent Gazette No. 508219/93)(SEQ ID NO. 6). Where the antigenic protein results from does not matter, so long as it has HCV antigenic activity. Natural isolates, chemical synthetics, and genetic recombination products can be used. Of the proteins in these regions, a peptide of varying length can be used as the antigenic protein. Preferably, a peptide composed of 8 or more amino acids containing at least one epitope is used. More preferably, a synthetic peptide having a molecular weight of 1,000 to 5,000 is used. The peptide can be synthesized by a known method in the art, such as solid phase synthesis, fragment condensation, or classical solution synthesis. Preferably, it can be produced by the solid phase peptide synthesis method described in the literature (Merrifield, J. Am. Chem. Soc. 85:2149, 1963). According to the present invention, one or more of core, NS3, NS4 and NS5 antigen proteins containing one or more different epitopes are combined, and can be used directly, or after conjugation to a carrier protein, to sensitize carrier particles. In the Examples to be described later on, a peptide containing the 49th to 68th (SEQ ID NO. 6) amino acids in the core region described in Officially Published Patent Gazette No. 508219/93 is used as the core antigen, a peptide containing the 1706th to 1725th (SEQ ID NO. 8), 1718th to 1737th (SEQ ID NO. 9), and 1724th to 1743rd (SEQ ID NO. 10) amino acids in the NS4 region described in Officially Published Patent Gazette No. 508219/93 is used as the NS4 peptide, a peptide containing the 2287th to 2306th (SEQ ID NO. 11), 2299th to 2318th (SEQ ID NO. 12), and 2311th to 2330th (SEQ ID NO. 13) amino acids in the NS5 region described in Officially Published Patent Gazette No. 508219/93 is used as the NS5 peptide, and a peptide containing the 1192nd to 1457th (SEQ ID NO. 7) amino acids in the NS3 region described in Officially Published Patent Gazette No. 508219/93 is used as the NS3 peptide. However, the antigenic proteins of the present invention are not restricted to these peptides.
Each of the above-described antigenic proteins is chemically bonded to the carrier protein to prepare a conjugated antigen. The antigenic protein has been found to show higher detection sensitivity when sensitizing the carrier particles as a conjugated antigen than when sensitizing them directly. For the antigen protein with a molecular weight of 10,000 or more like the NS3 antigen used in the present invention, however, no marked difference in the sensitivity has been observed. Thus, it is permissible to sensitize the carrier particles directly with such a high molecular weight antigen protein, and sensitize the particles with the other antigen proteins as conjugated antigens. Bonding of the carrier protein and the antigen protein can be performed by a known method using carbodiimide, periodic acid, maleimide or glutaraldehyde. The use of the glutaraldehyde method is preferred, because their bonding by glutaraldehyde-induced crosslinking increases reactivity. For the preparation of the conjugated antigen, the carrier protein and the antigen protein are mixed at a ratio, as the ratio of the numbers of molecules for the two, of about 1:3 to 1:20, preferably about 1:4 to 1:9, more preferably about 1:6 to 1:8. The so prepared conjugated antigen is bound to(or caused to sensitize) carrier particles by a known method, which may be, say, physical adsorption or chemical adsorption. As described previously, the NS3 antigen may be directly caused to sensitize carrier particles without forming a conjugated antigen together with the carrier protein. This can be performed by the same method as described above. Sensitization is carried out in a buffer, a solution with a buffer action, such as phosphate buffer, glycine buffer, TRIS buffer or acetate buffer, preferably at a pH of 3 to 8, more preferably at pH 4 to 5.
The present invention also provides a method of diagnosing hepatitis C virus infection, which comprises adding the aforementioned diagnostic reagent for hepatitis C virus infection to a sample, and measuring the degree of agglutination of the carrier particles by a flow cytometer. The diagnostic reagent for hepatitis C virus infection according to the present invention also reacts with anti-HCV antibodies, if present in the sample, to cause agglutination. The resulting agglutination may be measured visually or by turbidity or absorbance. However, a rapid, high-sensitivity, high-precision/accuracy measurement can be made by optically measuring the agglutinated particles with a full-automatic immunoagglutination measuring system (e.g., PAMIA-30(trademark), TOA MEDICAL ELECTRONICS Co., Ltd) which relies on the principle of a flow cytometer. In detail, the sample is guided into a flow cell, arranged in a row, and passed by a sheath flow mechanism. A laser beam is projected onto it, and the intensity of scattered light produced is measured to tell the degree of agglutination. The number of the agglutinated particles (P: Polymer) and the number of non-agglutinated particles (M: Monomer) are counted. From the P and M, P/T (T=P+M) is calculated, and the presence or absence of anti-HCV antibodies is qualitatively evaluated based on the cutoff value obtained beforehand. This method enables anti-HCV antibodies to be detected with high sensitivity. A suitable particle size of the particles would make measurement possible by a blood analyzer or a particle size analyzer using electrical resistance. However, measurement by an optical method is preferred to avoid a problem such as clogging of the detector.
The use of the diagnostic reagent for hepatitis C virus infection according to the present invention permits highly sensitive, early diagnosis of infection with hepatitis C virus as compared with commercially available diagnostic reagents. Actually, the diagnostic reagent of the present invention was tested using panel serum composed of several samples taken over time from the same individual in the course of seroconversion of anti-HCV antibodies (e.g., HCV Seroconversion Panel, imported and distributed by Kyowa Medics Co., Ltd, manufactured by BOSTON BIOMEDICA, INC.). The diagnostic reagent for hepatitis C virus infection of the present invention was demonstrated to detect HCV infection in an earlier stage than conventional methods, passive hemagglutination (PHA) using erythrocytes, enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA), namely, in the initial stage of infection.
The diagnostic reagent of the present invention was also compared with a diagnostic reagent produced by using the same antigen as in the inventive diagnostic reagent, but directly causing this antigen to sensitize carrier particles without preparing its conjugated antigen together with a carrier protein. The diagnostic reagent of the present invention was found to be superior in the detection sensitivity.
Furthermore, the diagnostic reagent of the present invention does not decrease in the detection sensitivity even after long-term storage. Thus, it proves a stable diagnostic reagent.
The diagnostic method of the present invention, compared with a conventional method such as ELISA, does not involve a complicated washing step, but can be performed by a mere step of mixing the inventive diagnostic reagent for HCV infection (preferably, latex particles sensitized with HCV antigen) with a sample (preferably, a subject""s blood). The diagnostic method of the present invention also enables measurement by a measuring system which performs both of the above mixing step and the measuring step full-automatically. Thus, this method is suitable for measuring many samples.